J Cancer 2017; 8(10):1774-1785. doi:10.7150/jca.19732

Research Paper

Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma

Makoto Nishino1#, Mari Ozaki1#, Ahmed E. Hegab1✉, Junko Hamamoto1, Shizuko Kagawa1, Daisuke Arai1, Hiroyuki Yasuda1, Katsuhiko Naoki1, Kenzo Soejima1, Hideyuki Saya2, Tomoko Betsuyaku1

1. Division of Pulmonary Medicine, Department of Medicine, and
2. Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
#These authors contributed equally.
†This work was supported by JSPS Grant-in-Aid for Scientific Research (A.E.H. and T.B.)

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Nishino M, Ozaki M, Hegab AE, Hamamoto J, Kagawa S, Arai D, Yasuda H, Naoki K, Soejima K, Saya H, Betsuyaku T. Variant CD44 expression is enriching for a cell population with cancer stem cell-like characteristics in human lung adenocarcinoma. J Cancer 2017; 8(10):1774-1785. doi:10.7150/jca.19732. Available from http://www.jcancer.org/v08p1774.htm

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Background: Preliminary studies have identified cancer stem cells (CSCs) in various cancers and there are several ongoing clinical studies targeting these cells. CD44 (standard or variant isoforms) and/or aldehyde dehydrogenase (ALDH) expression is the most commonly used markers for the identification of CSCs. The goal of the current study was to examine the ability of CD44v, either alone or in combination with ALDH, to identify CSCs within human lung cancer cells lines.

Methods: We examined several lung adenocarcinoma cell lines for the ability of CD44v and/or ALDH expression to enrich for cells with CSC characteristics such as in vitro differential proliferation rate, chemotherapeutic-resistance, tumorsphere formation, and in vivo tumorigenicity. We also compared their in vivo secondary tumor formation, and histological characteristics of their xenograft tumors, and examined their expression of PD-L1, EGFR, xCT, and reactive oxygen species (ROS).

Results: Both CD44vhigh/ALDHhigh and CD44vhigh/ALDHlow cells were enriched in cells with CSC characteristics, with the CD44vhigh/ALDHlow cells being more proliferative and more resistant to chemotherapeutics, whereas CD44vhigh/ALDHhigh cells were more efficient in forming tumorspheres in vitro, in making primary xenograft tumors, and in propagating secondary tumors in vivo. Applying stricter sorting gates to select for cells with the highest CD44v/ALDH expression caused the CD44vhigh/ALDHlow cells to lose their high proliferation rates and chemotherapeutic resistance ability, but enriched for the tumorsphere-forming cells among the CD44vhigh/ALDHhigh and CD44vhigh/ALDHlow cells. CD44vhigh expression was associated with PD-L1 and xCT expression in both H1650 and HCC827 cells. This association was not modified by ALDH expression in the H1650 cell line. However, in the HCC827 cell line, ALDH expression was negatively associated with PD-L1 and positively associated with xCT expression.

Conclusion: Lung adenocarcinoma cells with high CD44v expression are enriched for CSCs. Addition of ALDH as an enrichment marker bestowed some CSCs characteristics to CD44vhigh/ALDHlow cells and others to CD44vhigh/ALDHhigh cells. We propose that lung adenocarcinoma contains different CSCs, each of them endowed with different CSC characteristics.